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1.
Food Chem ; 449: 139220, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-38579657

RESUMEN

A colorimetry/fluorescence dual-mode assay based on the aptamer-functionalized magnetic covalent organic framework-supported CuO and Au NPs (MCOF-CuO/Au@apt) was developed for Salmonella typhimurium (S. typhimurium) biosensing. The nanohybrid combined three functions in one: good magnetic separation characteristic, excellent oxidase-mimic activity for tetrap-aminophenylethylene (TPE-4A), and target recognition capability. The attachment of MCOF-CuO/Au@apt onto the surface of S. typhimurium resulted in a significant reduction in the oxidase-mimicking activity of the nanohybrid, which could generate dual-signal of colorimetry and fluorescence through the catalytic oxidation of TPE-4A. Based on this, S. typhimurium could be specifically detected in the linear ranges of 102- 106 CFU·mL-1 and 101- 106 CFU·mL-1, with LODs of 7.6 and 2.1 CFU·mL-1, respectively in colorimetry/fluorescence modes. Moreover, the smartphone and linear discrimination analysis-based system could be used for on-site and portable testing. In addition, this platform showed applicability in detecting S. typhimurium in milk, egg liquid and chicken samples.


Asunto(s)
Técnicas Biosensibles , Colorimetría , Salmonella typhimurium , Salmonella typhimurium/aislamiento & purificación , Salmonella typhimurium/enzimología , Animales , Técnicas Biosensibles/instrumentación , Leche/microbiología , Leche/química , Fluorescencia , Pollos , Oro/química , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Contaminación de Alimentos/análisis , Nanopartículas del Metal/química , Espectrometría de Fluorescencia , Huevos/análisis , Huevos/microbiología
2.
J Mol Biol ; 434(12): 167598, 2022 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-35461877

RESUMEN

Cation selectivity and coupling are important attributes of cation-coupled symporters. Salmonella typhimurium melibiose permease (MelBSt) catalyzes the co-transport of galactosides with cations (H+, Li+, or Na+). 3-D crystal structures of MelBSt have revealed the molecular recognition for sugar substrates, but the cation binding and coupling mechanisms have not been defined to atomic levels. In its human homolog MFSD2A, a lethal mutation was mapped at its Na+-binding pocket; however, none of the structures in this subfamily resolved its cation binding. In this study, molecular dynamics simulations reveal the binding interactions of Na+ and Li+ with MelBSt. Interestingly, Thr121, the lethal mutation position in MFSD2A, forms stable interaction with Na+ but is at a distance from Li+. Most mutations among 11 single-site Thr121 mutants of MelBSt exhibited little effects on the galactoside binding, but largely altered the cation selectivity with severe inhibitions on Na+ binding. Few mutants (Pro and Ala) completely lost the Na+ binding and Na+-coupled transport, but their Li+ or H+ modes of activity were largely retained. It can be concluded that Thr121 is necessary for Na+ binding, but not required for the binding of H+ or Li+, so a subset of the Na+-binding pocket is enough for Li+ binding. In addition, the protein stability for some mutants can be only retained in the presence of Li+, but not by Na+ due to the lack of affinity. This finding, together with other identified thermostable mutants, supports that the charge balance of the cation-binding site plays an important role in MelBSt protein stability.


Asunto(s)
Proteínas Bacterianas , Salmonella typhimurium , Simportadores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cationes/metabolismo , Humanos , Litio/metabolismo , Melibiosa/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Sodio/metabolismo , Simportadores/química , Simportadores/genética , Simportadores/metabolismo
3.
J Bacteriol ; 204(1): e0020821, 2022 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-34662237

RESUMEN

Organismal adaptations to environmental stimuli are governed by intracellular signaling molecules such as nucleotide second messengers. Recent studies have identified functional roles for the noncanonical 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) in both eukaryotes and prokaryotes. In Escherichia coli, 2',3'-cNMPs are produced by RNase I-catalyzed RNA degradation, and these cyclic nucleotides modulate biofilm formation through unknown mechanisms. The present work dissects cellular processes in E. coli and Salmonella enterica serovar Typhimurium that are modulated by 2',3'-cNMPs through the development of cell-permeable 2',3'-cNMP analogs and a 2',3'-cyclic nucleotide phosphodiesterase. Utilization of these chemical and enzymatic tools, in conjunction with phenotypic and transcriptomic investigations, identified pathways regulated by 2',3'-cNMPs, including flagellar motility and biofilm formation, and by oligoribonucleotides with 3'-terminal 2',3'-cyclic phosphates, including responses to cellular stress. Furthermore, interrogation of metabolomic and organismal databases has identified 2',3'-cNMPs in numerous organisms and homologs of the E. coli metabolic proteins that are involved in key eukaryotic pathways. Thus, the present work provides key insights into the roles of these understudied facets of nucleotide metabolism and signaling in prokaryotic physiology and suggest broad roles for 2',3'-cNMPs among bacteria and eukaryotes. IMPORTANCE Bacteria adapt to environmental challenges by producing intracellular signaling molecules that control downstream pathways and alter cellular processes for survival. Nucleotide second messengers serve to transduce extracellular signals and regulate a wide array of intracellular pathways. Recently, 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) were identified as contributing to the regulation of cellular pathways in eukaryotes and prokaryotes. In this study, we define previously unknown cell processes that are affected by fluctuating 2',3'-cNMP levels or RNA oligomers with 2',3'-cyclic phosphate termini in E. coli and Salmonella Typhimurium, providing a framework for studying novel signaling networks in prokaryotes. Furthermore, we utilize metabolomics databases to identify additional prokaryotic and eukaryotic species that generate 2',3'-cNMPs as a resource for future studies.


Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica/fisiología , Nucleótidos Cíclicos/metabolismo , Salmonella typhimurium/enzimología , Proteínas Bacterianas/genética , Biopelículas , Endorribonucleasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos , Respuesta al Choque Térmico , Peróxido de Hidrógeno , Operón , ARN Bacteriano , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
4.
Cell Rep ; 37(12): 110130, 2021 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-34936863

RESUMEN

Bacterial toxin-antitoxin modules contribute to the stress adaptation, persistence, and dormancy of bacteria for survival under environmental stresses and are involved in bacterial pathogenesis. In Salmonella Typhimurium, the Gcn5-related N-acetyltransferase toxin TacT reportedly acetylates the α-amino groups of the aminoacyl moieties of several aminoacyl-tRNAs, inhibits protein synthesis, and promotes persister formation during the infection of macrophages. Here, we show that TacT exclusively acetylates Gly-tRNAGlyin vivo and in vitro. The crystal structure of the TacT:acetyl-Gly-tRNAGly complex and the biochemical analysis reveal that TacT specifically recognizes the discriminator U73 and G71 in tRNAGly, a combination that is only found in tRNAGly isoacceptors, and discriminates tRNAGly from other tRNA species. Thus, TacT is a Gly-tRNAGly-specific acetyltransferase toxin. The molecular basis of the specific aminoacyl-tRNA acetylation by TacT provides advanced information for the design of drugs targeting Salmonella.


Asunto(s)
Acetiltransferasas/metabolismo , Toxinas Bacterianas/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia de Glicerina/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Acetilación , Acetiltransferasas/química , Antitoxinas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , ADN Bacteriano , Procesamiento Proteico-Postraduccional , Infecciones por Salmonella/microbiología , Salmonella typhimurium/química
5.
mBio ; 12(6): e0310621, 2021 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-34809457

RESUMEN

Methylesterase/deamidase CheB is a key component of bacterial chemotaxis systems. It is also a prominent example of a two-component response regulator in which the effector domain is an enzyme. Like other response regulators, CheB is activated by phosphorylation of an aspartyl residue in its regulatory domain, creating an open conformation between its two domains. Studies of CheB in Escherichia coli and related organisms have shown that its enzymatic action is also enhanced by a pentapeptide-binding site for the enzyme at the chemoreceptor carboxyl terminus. Related carboxyl-terminal pentapeptides are found on >25,000 chemoreceptor sequences distributed across 11 bacterial phyla and many bacterial species, in which they presumably play similar roles. Yet, little is known about the interrelationship of CheB phosphorylation, pentapeptide binding, and interactions with its substrate methylesters and amides on the body of the chemoreceptor. We investigated by characterizing the binding kinetics of CheB to Nanodisc-inserted chemoreceptor dimers. The resulting kinetic and thermodynamic constants revealed a synergy between CheB phosphorylation and pentapeptide binding in which a phosphorylation mimic enhanced pentapeptide binding, and the pentapeptide served not only as a high-affinity tether for CheB but also selected the activated conformation of the enzyme. The basis of this selection was revealed by molecular modeling that predicted a pentapeptide-binding site on CheB which existed only in the open, activated enzyme. Recruitment of activated enzyme by selective tethering represents a previously unappreciated strategy for regulating response regulator action, one that may well occur in other two-component systems. IMPORTANCE Two-component signal transduction systems are a primary means by which bacteria sense and respond to their environment. Response regulators are key components of these systems. Phosphorylation of response regulators by cognate histidine kinases generate active conformations which act on specific targets, DNA sequences or proteins. The targets have been considered passive in this process. Our characterization of interaction between response regulator CheB and its target chemoreceptor revealed active participation of the target in response regulator action. We found that a pentapeptide sequence at the carboxyl terminus of Escherichia coli chemoreceptors is a selective tether that binds only phosphorylated CheB, thus selecting the form of this two-component enzyme active for covalent modification of the selecting chemoreceptor. Analogous pentapeptides are found on chemoreceptors in many bacterial species and are presumably also selective tethers. There may well be other, uncharacterized examples of active participation of target molecules in response to regulator action.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Escherichia coli/enzimología , Salmonella typhimurium/enzimología , Proteínas Bacterianas/genética , Sitios de Unión , Hidrolasas de Éster Carboxílico/genética , Quimiotaxis , Dimerización , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Péptidos/química , Péptidos/metabolismo , Fosforilación , Unión Proteica , Dominios Proteicos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Salmonella typhimurium/química , Salmonella typhimurium/genética
6.
Biochemistry ; 60(42): 3173-3186, 2021 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-34595921

RESUMEN

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5'-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and ß-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3'-phosphate and l-Ser to l-Trp. In this process, the roles played by ß-site residues proximal to the PLP cofactor have not yet been fully established. ßGln114 is one such residue. To explore the roles played by ßQ114, we conducted a detailed investigation of the ßQ114A mutation on the structure and function of tryptophan synthase. Initial steady-state kinetic and static ultraviolet-visible spectroscopic analyses showed the Q to A mutation impairs catalytic activity and alters the stabilities of intermediates in the ß-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and ßQ114A mutant enzymes. These comparisons establish that the protein structural changes are limited to the Gln to Ala replacement, the loss of hydrogen bonds among the side chains of ßGln114, ßAsn145, and ßArg148, and the inclusion of waters in the cavity created by substitution of the smaller Ala side chain. Because the conformations of the open and closed allosteric states are not changed by the mutation, we hypothesize that the altered properties arise from the lost hydrogen bonds that alter the relative stabilities of the open (ßT state) and closed (ßR state) conformations of the ß-subunit and consequently alter the distribution of intermediates along the ß-subunit catalytic path.


Asunto(s)
Proteínas Bacterianas/química , Triptófano Sintasa/química , Regulación Alostérica/genética , Proteínas Bacterianas/genética , Biocatálisis , Cinética , Mutagénesis Sitio-Dirigida , Mutación , Salmonella typhimurium/enzimología , Triptófano Sintasa/genética
7.
JCI Insight ; 6(23)2021 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-34710062

RESUMEN

Bacterial cancer therapy (BCT) shows great promise for treatment of solid tumors, yet basic mechanisms of bacterial-induced tumor suppression remain undefined. Attenuated strains of Salmonella enterica serovar Typhimurium (STm) have commonly been used in mouse models of BCT in xenograft and orthotopic transplant cancer models. We aimed to better understand the tumor epithelium-targeted mechanisms of BCT by using autochthonous mouse models of intestinal cancer and tumor organoid cultures to assess the effectiveness and consequences of oral treatment with aromatase A-deficient STm (STmΔaroA). STmΔaroA delivered by oral gavage significantly reduced tumor burden and tumor load in both a colitis-associated colorectal cancer (CAC) model and in a spontaneous Apcmin/+ intestinal cancer model. STmΔaroA colonization of tumors caused alterations in transcription of mRNAs associated with tumor stemness, epithelial-mesenchymal transition, and cell cycle. Metabolomic analysis of tumors demonstrated alteration in the metabolic environment of STmΔaroA-treated tumors, suggesting that STmΔaroA imposes metabolic competition on the tumor. Use of tumor organoid cultures in vitro recapitulated effects seen on tumor stemness, mesenchymal markers, and altered metabolome. Furthermore, live STmΔaroA was required, demonstrating active mechanisms including metabolite usage. We have demonstrated that oral BCT is efficacious in autochthonous intestinal cancer models, that BCT imposes metabolic competition, and that BCT has direct effects on the tumor epithelium affecting tumor stem cells.


Asunto(s)
Terapia Biológica , Neoplasias Colorrectales/terapia , Salmonella typhimurium/fisiología , Administración Oral , Animales , Aromatasa/metabolismo , Modelos Animales de Enfermedad , Epitelio , Ratones , Organoides , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética
8.
Nucleic Acids Res ; 49(20): 11800-11809, 2021 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-34581811

RESUMEN

High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the ß-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection.


Asunto(s)
Proteínas Bacterianas/química , Estrés Oxidativo , Fenilalanina-ARNt Ligasa/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Fenilalanina-ARNt Ligasa/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/ultraestructura
9.
mBio ; 12(5): e0209921, 2021 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-34544273

RESUMEN

The two-component system PhoP/PhoQ is essential for Salmonella enterica serovar Typhimurium virulence. Here, we report that PhoP is methylated extensively. Two consecutive glutamate (E) and aspartate (D)/E residues, i.e., E8/D9 and E107/E108, and arginine (R) 112 can be methylated. Individual mutation of these above-mentioned residues caused impaired phosphorylation and dimerization or DNA-binding ability of PhoP to a different extent and led to attenuated bacterial virulence. With the help of specific antibodies recognizing methylated E8 and monomethylated R112, we found that the methylation levels of E8 or R112 decreased dramatically when bacteria encountered low magnesium, acidic pH, or phagocytosis by macrophages, under which PhoP can be activated. Furthermore, CheR, a bacterial chemotaxis methyltransferase, was identified to methylate R112. Overexpression of cheR decreased PhoP activity but increased PhoP stability. Together, the current study reveals that methylation plays an important role in regulating PhoP activities in response to environmental cues and, consequently, modulates Salmonella virulence. IMPORTANCE Posttranslational modifications (PTMs) play an important role in regulating enzyme activities, protein-protein interactions, or DNA-protein recognition and, consequently, modulate many biological functions. We demonstrated that PhoP, the response regulator of PhoP/PhoQ two-component system, could be methylated on several evolutionally conserved amino acid residues. These amino acid residues were crucial for PhoP phosphorylation or dimerization, DNA-binding ability of PhoP, and Salmonella virulence. Interestingly, methylation negatively regulated the activity of PhoP. A bacterial chemotaxis methyltransferase CheR was involved in PhoP methylation. Methylation of PhoP could stabilize it in an inactive conformation. Our work provides a more informative depiction of PhoP PTM and markedly improves our understanding of the coordinate regulation of bacterial chemotaxis and virulence.


Asunto(s)
Proteínas Bacterianas/metabolismo , Metiltransferasas/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/enzimología , Salmonella typhimurium/patogenicidad , Animales , Proteínas Bacterianas/genética , Femenino , Regulación Bacteriana de la Expresión Génica , Humanos , Metilación , Metiltransferasas/genética , Ratones , Ratones Endogámicos BALB C , Salmonella typhimurium/genética , Virulencia
10.
Biomolecules ; 11(8)2021 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-34439822

RESUMEN

The genome of the halophilic archaea Haloferax mediterranei contains three ORFs that show homology with glutamine synthetase (GS) (glnA-1, glnA-2, and glnA-3). Previous studies have focused on the role of GlnA-1, suggesting that proteins GlnA-2 and GlnA-3 could play a different role to that of GS. Glutamine synthetase (EC 6.3.1.2) belongs to the class of ligases, including 20 subclasses of other different enzymes, such as aspartate-ammonia ligase (EC 6.3.1.1), glutamate-ethylamine ligase (EC 6.3.1.6), and glutamate-putrescine ligase (EC 6.3.1.11). The reaction catalyzed by glutamate-putrescine ligase is comparable to the reaction catalyzed by glutamine synthetase (GS). Both enzymes can bind a glutamate molecule to an amino group: ammonium (GS) or putrescine (glutamate-putrescine ligase). In addition, they present the characteristic catalytic domain of GS, showing significant similarities in their structure. Although these proteins are annotated as GS, the bioinformatics and experimental results obtained in this work indicate that the GlnA-2 protein (HFX_1688) is a glutamate-putrescine ligase, involved in polyamine catabolism. The most significant results are those related to glutamate-putrescine ligase's activity and the analysis of the transcriptional and translational expression of the glnA-2 gene in the presence of different nitrogen sources. This work confirms a new metabolic pathway in the Archaea domain which extends the knowledge regarding the utilization of alternative nitrogen sources in this domain.


Asunto(s)
Proteínas Arqueales/genética , Proteínas de Escherichia coli/genética , Regulación de la Expresión Génica Arqueal , Ácido Glutámico/metabolismo , Haloferax mediterranei/enzimología , Ligasas/genética , Fijación del Nitrógeno/genética , Putrescina/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Amoníaco/metabolismo , Proteínas Arqueales/metabolismo , Clonación Molecular , Biología Computacional/métodos , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Haloferax mediterranei/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Ligasas/metabolismo , Filogenia , Biosíntesis de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transcripción Genética
11.
J Chem Phys ; 154(17): 175101, 2021 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-34241057

RESUMEN

Electron paramagnetic resonance (EPR) spectroscopy is used to address the remarkable persistence of the native Arrhenius dependence of the 2-aminopropanol substrate radical rearrangement reaction in B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium from physiological to cryogenic (220 K) temperatures. Two-component TEMPOL spin probe mobility in the presence of 10 mM (0.08% v/v) 2-aminopropanol over 200-265 K demonstrates characteristic concentric aqueous-cosolvent mesodomain and protein-associated domain (PAD, hydration layer) solvent phases around EAL in the frozen solution. The mesodomain formed by the relatively small amount of 2-aminopropanol is highly confined, as shown by an elevated temperature for the order-disorder transition (ODT) in the PAD (230-235 K) and large activation energy for TEMPOL rotation. Addition of 2% v/v dimethylsulfoxide expands the mesodomain, partially relieves PAD confinement, and leads to an ODT at 205-210 K. The ODT is also manifested as a deviation of the temperature-dependence of the EPR amplitude of cob(II)alamin and the substrate radical, bound in the enzyme active site, from Curie law behavior. This is attributed to an increase in sample dielectric permittivity above the ODT at the microwave frequency of 9.5 GHz. The relatively high frequency dielectric response indicates an origin in coupled protein surface group-water fluctuations of the Johari-Goldstein ß type that span spatial scales of ∼0.1-10 Å on temporal scales of 10-10-10-7 s. The orthogonal EPR spin probe rotational mobility and solvent dielectric measurements characterize features of EAL protein-solvent dynamical coupling and reveal that excess substrate acts as a fluidizing cryosolvent to enable native enzyme reactivity at cryogenic temperatures.


Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón , Etanolamina Amoníaco-Liasa/química , Temperatura , Etanolamina Amoníaco-Liasa/metabolismo , Microondas , Salmonella typhimurium/enzimología , Solventes/química , Solventes/metabolismo
12.
J Bacteriol ; 203(14): e0014321, 2021 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-33941609

RESUMEN

Proteolysis is a fundamental property of all living cells. In the bacterium Salmonella enterica serovar Typhimurium, the HspQ protein controls the specificities of the Lon and ClpAP proteases. Upon acetylation, HspQ stops being a Lon substrate and no longer enhances proteolysis of the Lon substrate Hha. The accumulated HspQ protein binds to the protease adaptor ClpS, hindering proteolysis of ClpS-dependent substrates of ClpAP, such as Oat, a promoter of antibiotic persistence. HspQ is acetylated by the protein acetyltransferase Pat from acetyl coenzyme A (acetyl-CoA) bound to the acetyl-CoA binding protein Qad. We now report that low cytoplasmic Mg2+ promotes qad expression, which protects substrates of Lon and ClpSAP by increasing HspQ amounts. The qad promoter is activated by PhoP, a regulatory protein highly activated in low cytoplasmic Mg2+ that also represses clpS transcription. Both the qad gene and PhoP repression of the clpS promoter are necessary for antibiotic persistence. PhoP also promotes qad transcription in Escherichia coli, which shares a similar PhoP box in the qad promoter region with S. Typhimurium, Salmonella bongori, and Enterobacter cloacae. Our findings identify cytoplasmic Mg2+ and the PhoP protein as critical regulators of protease specificity in multiple enteric bacteria. IMPORTANCE The bacterium Salmonella enterica serovar Typhimurium narrows down the spectrum of substrates degraded by the proteases Lon and ClpAP in response to low cytoplasmic Mg2+, a condition that decreases protein synthesis. This control is exerted by PhoP, a transcriptional regulator activated in low cytoplasmic Mg2+ that governs proteostasis and is conserved in enteric bacteria. The uncovered mechanism enables bacteria to control the abundance of preexisting proteins.


Asunto(s)
Citoplasma/metabolismo , Magnesio/metabolismo , Proteasa La/metabolismo , Salmonella typhimurium/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Citoplasma/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteasa La/química , Proteasa La/genética , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidad por Sustrato
13.
Biochemistry ; 60(3): 231-244, 2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33428374

RESUMEN

We have examined the reaction of Salmonella enterica serovar typhimurium tryptophan (Trp) synthase α2ß2 complex with l-Trp, d-Trp, oxindolyl-l-alanine (OIA), and dioxindolyl-l-alanine (DOA) in the presence of disodium (dl)-α-glycerol phosphate (GP), using stopped-flow spectrophotometry and X-ray crystallography. All structures contained the d-isomer of GP bound at the α-active site. (3S)-OIA reacts with the pyridoxal-5'-phosphate (PLP) of Trp synthase to form a mixture of external aldimine and quinonoid complexes. The α-carboxylate of OIA rotates about 90° to become planar with the PLP when the quinonoid complex is formed, resulting in a conformational change in the loop of residues 110-115. The COMM domain of the Trp synthase-OIA complex is found as a mixture of two conformations. The (3R)-diastereomer of DOA binds about 5-fold more tightly than (3S)-OIA and also forms a mixture of aldimine and quinonoid complexes. DOA forms an additional H-bond between the 3-OH of DOA and ßLys-87. l-Trp does not form a covalent complex with the PLP of Trp synthase. However, d-Trp forms a mixture of two external aldimine complexes which differ in the orientation of the α-carboxylate. In one conformation, the α-carboxylate is in the plane of the PLP, while in the other conformation, the α-carboxylate is perpendicular to the PLP plane. These results confirm that the stereochemistry of the transient indolenine quinonoid intermediate in the mechanism of Trp synthase is (3S) and demonstrate the linkage between aldimine and quinonoid reaction intermediates in the ß-active site and allosteric communications with the α-active site.


Asunto(s)
Proteínas Bacterianas/química , Salmonella typhimurium/enzimología , Triptófano Sintasa/química , Triptófano/química , Proteínas Bacterianas/antagonistas & inhibidores , Dominio Catalítico , Estructura Secundaria de Proteína , Triptófano Sintasa/antagonistas & inhibidores
14.
Bioorg Med Chem ; 30: 115898, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33388594

RESUMEN

The spread of antibiotic resistance within the ESKAPE group of human pathogenic bacteria poses severe challenges in the treatment of infections and maintenance of safe hospital environments. This motivates efforts to validate novel target proteins within these species that could be pursued as potential targets for antibiotic development. Genetic data suggest that the enzyme FabG, which is part of the bacterial fatty acid biosynthetic system FAS-II, is essential in several ESKAPE pathogens. FabG catalyzes the NADPH dependent reduction of 3-keto-acyl-ACP during fatty acid elongation, thus enabling lipid supply for production and maintenance of the cell envelope. Here we report on small-molecule screening on the FabG enzymes from A. baumannii and S. typhimurium to identify a set of µM inhibitors, with the most potent representative (1) demonstrating activity against six FabG-orthologues. A co-crystal structure with FabG from A. baumannii (PDB:6T65) confirms inhibitor binding at an allosteric site located in the subunit interface, as previously demonstrated for other sub-µM inhibitors of FabG from P. aeruginosa. We show that inhibitor binding distorts the oligomerization interface in the FabG tetramer and displaces crucial residues involved in the interaction with the co-substrate NADPH. These observations suggest a conserved allosteric site across the FabG family, which can be potentially targeted for interference with fatty acid biosynthesis in clinically relevant ESKAPE pathogens.


Asunto(s)
Acinetobacter baumannii/enzimología , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Pseudomonas aeruginosa/enzimología , Salmonella typhimurium/enzimología , Oxidorreductasas de Alcohol/metabolismo , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Estructura Molecular , Relación Estructura-Actividad
15.
mBio ; 11(6)2020 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-33203749

RESUMEN

Salmonella enterica serovar Typhimurium is an intracellular pathogen that elicits nitric oxide (NO·) production by host macrophages. NO· is a potent antimicrobial mediator with diverse targets, including protein thiols and metal centers. The mobilization of zinc from metalloproteins by NO· increases the availability of free intracellular zinc, which is detrimental to bacterial cells, but the precise mechanism of zinc cytotoxicity is uncertain. Here, we show that excess zinc results in the mismetallation of the essential iron-containing enzyme peptide deformylase (PDF), thereby diminishing its activity. PDF mismetallation is observed in zinc-treated bacteria lacking the zinc exporters ZntA and ZitB and is also observed during nitrosative stress, suggesting that NO·-mediated zinc mobilization results in PDF mismetallation. However, NO· also inhibits PDF directly by S-nitrosylating the metal-binding Cys90 residue. These observations identify PDF as an essential bacterial protein that is subject to both direct and indirect inactivation by NO·, providing a novel mechanism of zinc toxicity and NO·-mediated antibacterial activity.IMPORTANCE We have previously shown that the host-derived antimicrobial mediator nitric oxide (NO·) mobilizes zinc from bacterial metalloproteins. The present study demonstrates that NO· inactivates the essential iron-containing enzyme peptide deformylase, both by promoting its mismetallation by zinc and by directly modifying its metal-binding site. We explain how free intracellular zinc is detrimental for cells and reveal a new mechanism of NO·-mediated bacterial growth inhibition that is distinct from previously known targets.


Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antiinfecciosos/farmacología , Hierro/metabolismo , Óxido Nítrico/farmacología , Salmonella typhimurium/efectos de los fármacos , Zinc/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Homeostasis , Macrófagos/microbiología , Metaloproteínas/metabolismo , Mutación , Estrés Nitrosativo , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Zinc/toxicidad
16.
Adv Clin Exp Med ; 29(12): 1433-1441, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33245853

RESUMEN

BACKGROUND: Enolase is generally known as the glycolytic pathway enzyme present in the cytoplasm of eukaryotic cells and in some microorganisms. In human cells, it is also a component of cell surface membranes, where it functions as a human plasminogen receptor. OBJECTIVES: The study aimed to purify Salmonella enterica serovar Typhimurium cytosolic enolase and obtain the antibodies against this protein; to identify enolase on the surface of bacteria; and to find cross-reactivity and plasminogen binding properties. MATERIAL AND METHODS: Cytosolic enolase from S. Typhimurium was purified using a five-step preparation method. Anti-cytosolic enolase antibodies combined with scanning electron microscopy (SEM) allowed us to detect enolase on the surface of intact S. Typhimurium cells. The binding of plasminogen to surface enolase and the cross-reactivity of this protein with antibodies against human enolases were tested with western blot. RESULTS: Antibodies against human α- and ß-enolases cross-reacted with S. Typhimurium membrane protein, the identity of which was further confirmed using a mass spectrometry analysis of enolase tryptic peptides. The enolase form bacterial membrane also bound plasminogen. CONCLUSIONS: The cross-reactivity of membrane enolase with antibodies against human enolases suggests that this bacterium shares epitopes with human proteins. Surface exposition of enolase and the demonstrated affinity for human plasminogen indicates that Salmonella membrane enolase could play a role in the interaction of S. Typhimurium with host cells.


Asunto(s)
Plasminógeno , Salmonella typhimurium , Proteínas Portadoras , Humanos , Proteínas de la Membrana , Fosfopiruvato Hidratasa/metabolismo , Plasminógeno/metabolismo , Salmonella typhimurium/enzimología
17.
Biochemistry ; 59(48): 4573-4580, 2020 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-33231431

RESUMEN

Non-typhoidal Salmonella are capable of colonizing livestock and humans, where they can progressively cause disease. Previously, a library of targeted single-gene deletion mutants of Salmonella enterica serotype Typhimurium was inoculated to ligated ileal loops in calves to identify genes under selection. Of those genes identified, a cluster of genes is related to carbohydrate metabolism and transportation. It is proposed that an incoming carbohydrate is first phosphorylated by a phosphoenolpyruvate-dependent phosphotransferase system. The metabolite is further phosphorylated by the kinase STM3781 and then cleaved by the aldolase STM3780. STM3780 is functionally annotated as a class II fructose-bisphosphate aldolase. The aldolase was purified to homogeneity, and its aldol condensation activity with a range of aldehydes was determined. In the condensation reaction, STM3780 was shown to catalyze the abstraction of the pro-S hydrogen from C3 of dihydroxyacetone and subsequent formation of a carbon-carbon bond with S stereochemistry at C3 and R stereochemistry at C4. The best aldehyde substrate was identified as l-threouronate. Surprisingly, STM3780 was also shown to catalyze the condensation of two molecules of dihydroxyacetone phosphate to form the branched carbohydrate dendroketose bisphosphate.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Genes Bacterianos , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Animales , Biocatálisis , Metabolismo de los Hidratos de Carbono , Carbohidratos/química , Bovinos , Enfermedades de los Bovinos/microbiología , Medición de Intercambio de Deuterio , Dihidroxiacetona Fosfato/metabolismo , Humanos , Familia de Multigenes , Resonancia Magnética Nuclear Biomolecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonelosis Animal/microbiología , Serogrupo , Estereoisomerismo , Especificidad por Sustrato
18.
PLoS Genet ; 16(10): e1009085, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33125364

RESUMEN

DNA supercoiling is essential for all living cells because it controls all processes involving DNA. In bacteria, global DNA supercoiling results from the opposing activities of topoisomerase I, which relaxes DNA, and DNA gyrase, which compacts DNA. These enzymes are widely conserved, sharing >91% amino acid identity between the closely related species Escherichia coli and Salmonella enterica serovar Typhimurium. Why, then, do E. coli and Salmonella exhibit different DNA supercoiling when experiencing the same conditions? We now report that this surprising difference reflects disparate activation of their DNA gyrases by the polyamine spermidine and its precursor putrescine. In vitro, Salmonella DNA gyrase activity was sensitive to changes in putrescine concentration within the physiological range, whereas activity of the E. coli enzyme was not. In vivo, putrescine activated the Salmonella DNA gyrase and spermidine the E. coli enzyme. High extracellular Mg2+ decreased DNA supercoiling exclusively in Salmonella by reducing the putrescine concentration. Our results establish the basis for the differences in global DNA supercoiling between E. coli and Salmonella, define a signal transduction pathway regulating DNA supercoiling, and identify potential targets for antibacterial agents.


Asunto(s)
Girasa de ADN/genética , ADN-Topoisomerasas de Tipo I/genética , ADN Superhelicoidal/genética , Escherichia coli/genética , Salmonella typhimurium/genética , Girasa de ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/efectos de los fármacos , ADN Superhelicoidal/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Magnesio/farmacología , Putrescina/farmacología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/enzimología , Espermidina/biosíntesis
19.
J Vis Exp ; (163)2020 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-33044464

RESUMEN

Structural studies with tryptophan synthase (TS) bienzyme complex (α2ß2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated α- and isolated ß-subunit allowed the purification of high amounts of pure subunits and α2ß2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme ß-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α2ß2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α2ß2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the α2ß2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant α2ß2 StTS complexes.


Asunto(s)
Mutagénesis Sitio-Dirigida/métodos , Proteínas Mutantes/química , Proteínas Mutantes/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Triptófano Sintasa/genética , Triptófano Sintasa/aislamiento & purificación , Catálisis , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli/metabolismo , Subunidades de Proteína/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Electricidad Estática , Triptófano Sintasa/química
20.
World J Microbiol Biotechnol ; 36(11): 168, 2020 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-33029674

RESUMEN

Salmonella is a well-known food-borne pathogen causing disease in humans and animals worldwide. Peptidyl-prolyl isomerases (PPIases) catalyse the cis-trans isomerisation of prolyl bound, which is a slow and rate-limiting step of protein folding. Here, we present the biochemical and molecular characterisation of a novel multi-domain parvulin-type, PPIases-C from the pathogenic bacteria Salmonella Typhimurium, annotated as rPpiC. The recombinant plasmid PpiC_pET28c was used for protein induction using 1.5 mM concentration of isopropyl-ß-D-thiogalactopyranoside at 30 °C. Subsequently, the protein was identified by using the LC-MS technique showing high match score and sequence coverage with available PPIases-C proteins database. Using the succinyl-ala-phe-pro-phe-p nitroanilide as a substrate, Vmax of the enzyme was found to be 0.8187 ± 0.1352 µmoles/min and Km = 1.6014 ± 0.8449 µM, respectively. With this, we conclude that rPpiC protein is an active form of protein from Salmonella Typhimurium and plays an important role in protein folding.


Asunto(s)
Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Pliegue de Proteína , Salmonella typhimurium/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Especificidad por Sustrato
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